Transmissible gastroenteritis (TGE) is an economically important, acute enteric disease of swine, which is often 100% fatal in newborn piglets (Enjuanes, et al. (1995) Dev. Biol. Stand. 84:145-152; Enjuanes, et al. (1995) Adv. Exp. Med. Biol. 380:197-211; Laude, et al. (1990) Vet. Microbiol. 23:147-154). TGE virus (TGEV), the causative agent of TGE, is a member of the Coronaviridae family and the order Nidovirales. In addition to the Coronaviridae, the order Nidovirales also includes the Arteriviridae family, of which the swine pathogen porcine reproductive and respiratory syndrome virus (PRRSV) is a member (Cavanagh and Horzinek (1993) Arch. Virol. 128:395-396; de Vries, et al. (1997) Semin. Virol. 8:33-47; Siddell, et al. (1983) J. Gen. Virol. 64:761-776). Despite significant size differences (˜13 to 32 kb), the polycistronic genome organization and regulation of gene expression from a nested set of subgenomic mRNAs are similar for all members of the order (de Vries, et al. (1997) Semin. Virol. 8:33-47; Snijder and Horzinek (1993) J. Gen. Virol. 74:2305-2316).
TGEV possesses a single-stranded, positive-sense ˜28.5-kb RNA genome enclosed in a helical nucleocapsid structure that is surrounded by an envelope containing three viral proteins, including the S glycoprotein, the membrane (M) glycoprotein and a small envelope (E) protein (Eleouet, et al. (1995) Virology 206:817-822; Enjuanes and van der Zeijst (1995) In: S. G. Siddell (ed.), The Coronaviridae. Plenum Press, New York, N.Y., p. 337-376; Rasschaert and Laude (1987) J. Gen. Virol. 68:1883-1890; Risco, et al. (1996) J. Virol. 70:4773-4777). Remarkably, only the E and M proteins are absolutely required for particle formation, defining a novel model for virion budding (Fischer, et al. (1998) J. Virol. 72:7885-7894; Vennema, et al. (1996) EMBO J. 15:2020-2028). The TGEV genome contains eight large open reading frames (ORFs), which are expressed from full-length or subgenomic-length mRNAs during infection (Eleouet, et al. (1995) Virology 206:817-822; Sethna, et al. (1991) J. Virol. 65:320-325; Sethna, et al. (1989) Proc. Natl. Acad. Sci. USA 86:5626-5630). The 5′-most ˜20 kb contains the replicase genes in two ORFs, 1A and 1B, the latter of which is expressed by ribosomal frameshifting (Almazan, et al. (2000) Proc. Natl. Acad. Sci. USA 97:5516-5521; Eleouet, et al. (1995) Virology 206:817-822). The 3′-most ˜9 kb of the TGEV genome contains the structural genes, each preceded by a highly conserved transcription regulatory element (TSE) [ACTAAAC; SEQ ID NO:1]. The size of the functional TSE is subject to debate, but ranges from ˜7-15+ nucleotides in length when analyzed in recombinant defective interfering RNAs (Enjuanes, et al. (2001) J. Biotechnology 88:183-204; Jeong, et al. (1996) Virology 217:311-322; Krishnan, et al. (1996) Virology 218:400-405; Joo and Makino (1995) J. Virol. 69:3339-3346). In general, TSE length affects the function of individual mutations because longer elements are generally more resistant to “debilitating” mutations (Enjuanes, et al. (2001) J. Biotechnology 88:183-204). As the leader RNA sequence is also defined by a TSE at its 3′ end, some degree of base-pairing between the leader RNA and body TSE likely mediate virus transcription of subgenomic RNAs (Baker and Lai (1990) EMBO J. 9:4173-4179; Baric, et al. (1983) J. Virol. 48:633-640; Makino, et al. (1986) Proc. Natl. Acad. Sci. USA 83:4204-4208; Makino, et al. (1991) J. Virol. 65:6031-6041; Siddell, S. G. 1995. The coronaviridae, An introduction. In: The coronaviridae, eds. S. G. Siddell, Plenum Press, New York. pp 1-10). The subgenomic mRNAs are arranged in a co-terminal nested set structure from the 3′ end of the genome, and each contains a leader RNA sequence derived from the 5′ end of the genome. Although each mRNA is polycistronic, the 5′-most ORF is preferentially translated, necessitating the synthesis of a distinct mRNA species for each ORF (Lai and Cavanagh (1997) Adv. Virus Res. 48:1-100; McGoldrick, et al. (1999) Arch. Virol. 144:763-770; Sethna, et al. (1991) J. Virol. 65:320-325; Sethna, et al. (1989) Proc. Natl. Acad. Sci. USA 86:5626-5630). Both full-length and subgenomic-length negative-strand RNAs are also produced and have been implicated in mRNA synthesis (Baric and Yount (2000) J. Virol. 74:4039-4046; Sawicki and Sawicki (1990) J. Virol. 64:1050-1056; Schaad and Baric (1994) J. Virol. 68:8169-8179; Sethna, et al. (1991) J. Virol. 65:320-325; Sethna, et al. (1989) Proc. Natl. Acad. Sci. USA 86:5626-5630). Subgenomic RNA synthesis occurs by a method of discontinuous transcription, most likely by transcription attenuation during negative-strand synthesis (Baric and Yount (2000) J. Virol. 74:4039-4046; Sawicki and Sawicki (1990) J. Virol. 64:1050-1056).
The coronavirus E and M proteins function in virion assembly and release, which involve the constitutive secretory pathway of infected cells. Coexpression of the E and M proteins results in virus-like particle formation in cells, defining a novel, nucleocapsid-independent mechanism of enveloped-virus assembly (Vennema, et al. (1996) EMBO J. 15:2020-2028). The role of the E protein in virus assembly was further confirmed by reverse genetic analysis using targeted recombination (Fischer, et al. (1998) J. Virol. 72:7885-7894) and the development of TGEV replicon viruses (Curtis, et al. (2002) J. Virol. 76(3):1422-34). The TGEV M protein may serve to initiate the viral particle assembly process through interactions with genomic RNA and nucleoprotein in pre-Golgi compartments (Narayanan, et al. (2000) J. Virol. 74:8127-8134). The precise role of E in the assembly and release of coronavirus particles is not clear. Although an interaction between the E and M proteins has not yet been demonstrated, such an interaction likely occurs and would serve to facilitate the budding of viral particles. Additionally, E protein has been suggested to “pinch off the neck” of the assembled viral particles during the final stages of budding (Vennema, et al. (1996) EMBO J. 15:2020-2028).
PRRSV is endemic in most swine producing countries. Virions are enveloped 45-70 nm particles that contain 5 envelope proteins and an icosahedral nucleocapsid (N), which surrounds a single-stranded positive polarity RNA genome of about 15 kb (Pancholi, et al. (2000) J. Infect. Dis. 182:18-27; Pirzadeh and Dea (1998) J. Gen. Virol. 79:989-99). The 15 kDa N protein is most abundant and contains common conformational antigenic sites that are conserved of European and North American strains (Pirzadeh and Dea (1998) J. Gen. Virol. 79:989-99). N is likely multimerized to form icosahedral core structures (20-30 nm), which can be observed by EM. The major envelope proteins include a 25 kDa glycoprotein (GP5) and an 18-19 kDa unglycosylated M protein (Eleouet, et al. (1995) Virology 206:817-22; Meulenberg, et al. (1997) Vet. Microbiol. 55:197-202). GP5 (ORF 5) heterogeneity ranges from 50-90% amino acid identity among isolates, contains at least two neutralizing sites, and expression causes apoptosis (Eleouet, et al. (1995) Virology 206:817-22; Pirzadeh, et al. (1998) Can. J. Vet. Res. 62:170-7; Saif (1999) Transmissible gastroenteritis and porcine respiratory coronavirus, p. 295-325. In B. Straw, D'Allaire, S, Mengeling, W L and Taylor, D J (ed.), Diseases of Swine 8th edition. Iowa State University Press, Ames, Iowa; Tresnan, et al. (1996) J. Virol. 70:8669-74). The M protein (ORF6) contains 3 hydrophobic domains and accumulates in the ER of infected cells, where it forms disulfide-linked heterodimers with GP5 and may function in virus assembly (Meng (2000) Vet. Microbiol. 74:309-29). As with equine arterivirus, it is likely that coexpression of M and GP5 are needed for appropriate post-translational modification, folding and function, and for inducing high neutralizing antibody titers (Balasuriya, et al. (2000) J. Virol. 74:10623-30; Eleouet, et al. (1995) Virology 206:817-220).
Live, attenuated PRRSV vaccines causes viremia and may spread to other pigs. DNA immunization with a plasmid encoding GP5 of PRRSV induces specific neutralizing antibodies and reduces viremia and lung pathology in swine following challenge (Risco, et al. (1996) J. Virol. 70:4773-7). Recombinant adenovirus and vaccinia viruses encoding various PRRSV antigens are also being developed with encouraging results (Budzilowicz, et al. (1985) J. Virol. 53:834-40; Tresnan, et al. (1996) J. Virol. 70:8669-74). Several groups have concluded that effective PRRSV recombinant vaccines must induce high neutralizing titers, induce cellular immunity, induce heterotypic immunity and provide protection at mucosal surfaces (Eleouet, et al. (1995) Virology 206:817-22; Meulenberg, et al. (1997) Vet. Microbiol. 55:197-202). Achieving these goals is complicated by the generally low immunogenicity of the PRRSV envelope proteins and high genomic heterogeneity present in field isolates (Meulenberg, et al. (1997) Vet. Microbiol. 55:197-2020). Hence, improved vaccines are needed.
Recently, a simple and rapid approach for systematically assembling a full-length cDNA copy of the TGEV genomic RNA from which infectious transcripts can be produced has been described (Yount, et al. (2000) J. Virol. 74:10600-10611). This approach, as well as that of Almazan et al. ((2000) Proc. Natl. Acad. Sci. USA 97:5516-5521), will facilitate reverse genetic methods that impact all aspects of coronavirology, however, the production of infectious TGEV replicon particles is still limited.